Transgenic animals carry a gene which has been introduced into the germ line of an animal or an ancestor of the animal at an early stage in development. Wagner et al., Proc. Natl. Acad. Sci. USA, 78:5016 (1982). The ability to introduce new genes into the germ line of animals and thereby produce proteins outside of their normal environment and separated from their usual physiological control mechanisms has been extremely valuable for studying various aspects of gene expression. The technology also presents significant potential for improving various traits in animals, such as resistance to disease, reproductive rates and growth and lactation.
Much of the work involving transgens expression in animals has utilized the mouse as the experimental animal. For example, U.S. Pat. No. 4,736,866, describes the generation of transgenic mice whose germ line cells and somatic cells carry an activated oncogene sequence introduced into the animal or the ancestor of the animal at a germ line stage. Numerous other genes have been introduced into mice in an effort to gain a better understanding of gene expression in animals. See, for example, a review by Brinster and Palmiter, Harvery Lectures, 80:1-38 (1986) and Palmiter and Brinster, Annual Review of Genetics, 20:465-499 (1986).
More recently, gene transfer has been extended to domestic animals, including pigs, chickens, fish, cattle, rabbits and sheep. Pursel et al., Science, 244:1281-88 (1989). Many of the experiments in commercially important livestock have been designed to test the feasibility of introducing foreign growth promoting genes into the germ line of the livestock and thereby enhance growth performance. One approach that has received a great deal of attention is the introduction of growth regulating genes under the control of heterologous promoters into the germ line of the livestock, to allow long term production of the peptides in ectopic tissue.
The first attempt at applying the technology to domestic animals involved the introduction of a fusion gene including the mouse metallothionein (MT) regulatory/promoter sequence fused to the human growth hormone gene into the genome of pigs. Hammer et al., Nature, 315:380 (1985); Brem, Zuchygiene, 20:251 (1985). Since that time, several other growth promoting genes, including rat, ovine and bovine growth hormone, human growth releasing-factor and bovine insulin-like growth factor have been introduced into the germ lines of a variety of domestic animals. Most of the transferred genes in commercially important livestock have been under the control of the mouse MT promoter.
The foregoing strategy has resulted in the stimulation of growth and enhancement of conversion of food to protein in pigs, Pursel et al., Science, 244:1281 (1989), which indicates that an important practical utility for the technology exists. Unfortunately, the procedures heretofore employed have also resulted in detrimental side effects on the general health of the transgenic animals. Id.
In addition to the demand for improved rate and efficiency of body weight gain in commercially important livestock, there is also a strong demand in the agricultural industry for altered meat composition toward a leaner, less fat product consistent with medical advice that human beings reduce their consumption of animal fat. Although a side effect of the growth enhancing experimentation in transgenic animals has been a reduction in subcutaneous fat, the other deleterious side effects associated with the technology indicate that the prior technology is not a viable technique for providing leaner transgenic livestock. Other current strategies for growth regulation, including a shift toward leaner animals, include implantation or oral administration of natural or synthetic steroids, injection of exogenous somatotropin (growth hormone) or growth hormone releasing factor, oral or parenteral administration of B-adrenergic agonists and immunoneutralization. For a review, see Beerman, Status of Current Strategies For Growth Regulation in ANIMAL GROWTH REGULATION, 377 (1989).
While various of these techniques have been successfully utilized to reduce carcass fat in animals, each involves the routine administration of the factors to individual animals and does not result in alteration of the germ line to provide a continuous source of leaner animals. Moreover, hormonal treatments have an effect on many tissues, not just the tissue whose composition is to be altered. Alternative approaches for use of gene manipulation to alter body composition toward less fat tissue are therefore indicated.
One of the advances that would significantly increase the likelihood of success of producing leaner strains of transgenic animals would be to place the genetic material under the control of a promoter or enhancer sequence specific for fat (adipose) tissue. Use of such a tissue specific control element should result in expression of the genes only in the tissue whose composition is to be altered. Such an adipocyte-specific control element could be used, not only in the agricultural industry to produce animals with reduced fat content, but could also be utilized to alter fat metabolism in experimental animals. As an example, the fat-specific element could be used to alter the levels of endogenous genes that are thought to play key roles in the functioning of adipocytes, thereby allowing a better understanding of their roles in both adipose homeostasis and in disease states involving this tissue. The element could also be utilized in the development of small, organic pharmaceutical molecules which interfere with the binding of the protein factor to the adipose specific regulatory element, to thereby control transcription in a manner designed to combat regulatory defects associated with the disease state in fat tissue.
Prior research efforts have been directed to the elucidation of promoter/regulator sequences responsible for the expression of various proteins expressed primarily in adipocytes (fat-filled cells). For example, the promoter of the aP2 gene has been isolated and used as a model for the study of differentiation- and hormonally-linked gene regulation (Hunt et al., Proc. Natl. Acad. Sci. USA, 83:3786-3790 (1986); Phillips et al., J. Biol. Chem., 261: 10821-10827 (1986); Cook et al., Proc. Natl. Acad. Sci. USA, 85:2949-2953 (1988). AP2 is a novel gene product which is transcriptionally activated during adipocyte differentiation and is a member of the lipid-binding protein family.) Sequences from the aP2 proximal promoter (-247 or -168 to +21) have been shown to direct differentiation-dependent expression of the bacterial chloramphenicol acetyltransferase (CAT) upon transient transfection into preadipocytes and adipocytes (Distel et al., Cell, 49:835-844 (1987); Yang et al., Proc. Natl. Acad. Sci. USA, 86:3629-3633 (1989); Cook et al., Proc. Natl. Acad. Sci. USA, 85:2949-2953 (1988); Christy et al., Proc. Natl. Acad. Sci. USA, 86:3629-3633 (1989)) and several regulatory elements that strongly influence this expression have been identified. These include an AP-1 site at -120, where a sequence-specific interaction between Fos-containing protein complexes and DNA was first demonstrated (Distel et al., Cell, 49:835-844 (1987); Rauscher et al., Cell, 52:471-480 (1988)). An additional positive-acting element at position -140 was shown to bind the transcription factor C/EBP in extracts from adipose cells and a distinct protein from preadipocyte extracts (Christy et al., Proc. Natl. Acad. Sci. USA, 86:3629-3633 (1989); Herrera et al., Mol. Cell Biol., 9:5331-5339 (1989). Both the AP-1 and C/EBP binding sites have been shown to function positively in adipose cells and the AP-1 site is required for response of this promoter to cyclic AMP analogues (Herrera et al., Mol. Cell Biol., 9:5331-5339 (1989); Christy et al., Proc. Natl. Acad. Sci. USA, 86:3629-3633 (1989).
The promoter for the adipose-specific gene product, adipsin, has also been studied in some detail.
None of these sequences have heretofore been shown to direct expression of genetic material in fat tissue in the animal. The current inability to direct expression directly to adipose tissue in vivo continues to be a major problem that has hampered the use of transgenic technology to regulate the fat metabolism of domestic animals.
Accordingly, it is an object of the present invention to provide DNA sequences capable of directing the expression of recombinant proteins specifically in adipose tissue in vivo.
Another object of the present invention is to provide an expression system comprising the adipose-specific DNA sequence operatively linked to a DNA sequence coding for a protein capable of altering adipose tissue metabolism.
Another object of the present invention is to provide transgenic animals exhibiting altered fat tissue metabolism that can be used as animal models in the study of adipose homeostasis and disease states associated with fat tissue.
A still further object of the present invention is to provide transgenic livestock with decreased adipose tissue content which are leaner than non-transgenic animals of the same species.
Yet another object of the invention is to identify DNA sequences which, upon binding of a trans-acting protein factor, are primarily responsible for adipose differentiation-dependent expression of the gene.